Opalina. 201 
difference in clearness in the two nuclei. In individuals in which 
the system of excretory vacuoles’) is well developed, one sees that 
these vacuoles usually lie close along one side of the posterior 
nucleus (Fig. 1, Pl. XIV, 248, Pl. XXVI). They may extend also 
alongside of the anterior nucleus, though this is less usual. When 
such a vacuole is large and lies above the nucleus under observation, 
one is likely to see the nuclear structures very clearly. If, as 
sometimes occurs, the system of vacuoles divides, sending also a 
branch along the opposite side of the posterior nucleus, one has his 
best opportunity to observe this nucleus. if only the animal is so 
oriented. that one vacuole lies above and the other below the nucleus. 
In this case the refractive bodies in the cytoplasm (to be discribed 
later) lie so far above or below the focal plane of the objective of 
the microscope that they distort the image but little. It is therefore 
well to search for the most favorable individuals before settling 
down to careful study of the nucleus. The anterior part of the 
excretory organ is seldom well seen in the living animal. It is 
chiefly through the study of stained preparations that one reaches 
this explanation of the remarkable clearness of some living nuclei. 
In studying the reproductive processes in the spring, it is often 
valuable first to use living animals and later to treat the same 
animals with acetic acid or acetic carmine. For example, one can 
thus allow copulation to proceed to a particular point, and can 
then confirm his observations of nuclear and other phenomena in 
the living animals by studying the same animals treated with one 
of these reagents. It is often well in such cases to use first acetic 
acid and, after study, to follow with acetic carmine. Some structures, 
the nucleolus for example, show far better with acetic acid than 
with acetic carmine. Often the whole structure of cytoplasm and 
nucleus comes out with remarkable clearness with acetic acid. 
Acetic carmine used upon fresh material is very satisfactory 
for the study of the outlines of the excretory organs and is invaluable 
in the study of the minute forms in the spring, for, while it is not 
a sharply definitive stain and while its results, even in the same 
slide, are often very uneven, yet it is so simple in its application 
and so prompt in giving its results, that with it one can examine 
a very great amount of material, and this is essential to the proper 
understanding of the reproductive processes. 
Intra vitam staining with all the usual dyes was tried upon all 
") Metcaxr, 1907b and c. 
